Fusion polypeptides, and use thereof in antivascular tumor therapy

ABSTRACT

The present invention relates to fusion polypeptides, comprising at least two peptides. The invention further relates to the use of these fusion proteins in antivascular therapy of neoplastic diseases and to their use I the production of a drug for the treatment of neoplastic diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national phase application of InternationalApplication No. PCT/EP2004/009364, filed Aug. 20, 2004, which claimspriority to German Patent Application No. 103 38 733.1, filed Aug. 22,2003. The disclosures of these applications are hereby incorporated byreference in their entirety.

The present invention relates to fusion polypeptides, comprising atleast two peptides. One peptide comprises from 3 to 30 amino acids andpermits the fusion polypeptide to be bound selectively to endothelialcells in tumor vessels. The other peptide consists of the tissue factor(TF) or a fragment thereof, the tissue factor and the fragment beingcharacterized in that they are able to activate blood clotting uponbinding of the fusion polypeptide to endothelial cells in tumor vessels.The peptides can be joined together either directly or via a linkerhaving up to 15 amino acids. The invention further relates to the use ofthese fusion proteins in antivascular therapy of neoplastic diseases andto their use in the production of a drug for the treatment of neoplasticdiseases.

BACKGROUND OF THE INVENTION

Adequate neovascularization is a prerequisite for progressive tumorgrowth (1). Neoangiogenesis is required in particular for maintainingexpansive tumor growth, since only sufficient oxygenation will ensurethe supply with nutrients to and removal of tumor degradation productsfrom the tumor.

In the prior art directed to tumor treatment antivascular therapeuticstrategies have been developed, which are aimed at destruction of thetumor blood vessels and associated tumor infarction, in addition toanti-angiogenic therapeutic strategies, which attack the complex processof growth and differentiation of blood vessels.

A precondition for these strategies is identification of targetstructures in the vascular endothelium of the tumor that do not occur onresting endothelial cells in normal tissue. These specific targetstructures could be utilized in order to apply cytostatics or certaintoxins to the vascular endothelial cells of the tumor to a lesser extentto the tumor cells themselves. Target structures that can be used forthis purpose are bFGF (basic fibroblast growth factor), VEGF (vascularendothelial growth factor) and VEGFR-2 (VEGF receptor 2), endoglin,endosialin, a fibronectin isoform (ED-B domains), the integrins α_(v)β₃,α_(v)β₅, α₁β₁, and α₁β₂, aminopeptidase N, NG2 proteoglycan and thematrix metalloproteinases 2 and 9 (MMP 2 and 9) (2-13). For example,Arap et al. (8) coupled peptides that bind alpha1-integrinsspecifically, to an active substance that was being used in the state ofthe art for chemotherapy (doxorubicin). It was demonstrated in an animalmodel that the antineoplastic effect of doxorubicin could be improved bycoupling to the peptides.

An alternative antivascular therapeutic approach comprises selectiveactivation of blood clotting in tumor vessels, in order to induce tumornecrosis. For example, a bispecific F(ab′)2 antibody fragment wasproduced, which is directed against truncated tissue factor (tTF) and anMHC class II antigen. After experimental induction of the antigen intumor endothelial cells, an antivascular therapy could be demonstratedby administering the antibody in a murine neuroblastoma model (14). In asecond study by the same team, an immunoconjugate was used, whichcouples tTF selectively to a naturally occurring marker of the tumorvessel endothelium, VCAM-1 (vascular cell adhesion molecule-1) (15).

In a very similar approach, an antibody fragment (scFv), which isspecific for the oncofetal ED-B domain, was fused with tTF. The fusionproteins generated, scFv-tTF, led to a complete and selective infarctionin various tumors in the mouse model (16).

Alternatively, tTF was coupled to an inhibitor of the prostate-specificmembrane antigen (17). This fusion protein induced selective infarctionnecrosis in a rat prostate model after intravenous administration.Administering this fusion protein in combination with a cytotoxicsubstance (doxorubicin) at low dose resulted in massive tumor regressionand even complete tumor eradication (17). Other tTF fusion proteins,consisting of antibody fragments against VEGFR (VEGF receptor), endoglinand VCAM-1, have been described recently (18).

However, the molecules produced for antivascular tumor therapy in thestate of the art have drawbacks. In particular it has to be assumed thatthese molecules are immunogenic owing to their size. Treatment ofmammals with these molecules will therefore trigger an immune reactionagainst the molecules, so that repeated administration of the moleculesbecomes impossible.

The size of the coupling partner, by means of which the peptide portion,which can activate blood clotting, is to be directed onto the tumortissue, may further cause steric hindrance to a formation of themacromolecular factor VIIa/FX enzyme-substrate complex, which isimportant for blood clotting. Formation of the complex can also behampered when the peptide capable of activating blood clotting has analtered conformation owing to the relatively large fusion partners.

In the state of the art (WO 03/035688), fusion polypeptides are alsoknown wherein a selective binding domain, e.g. a domain of fibronectinthat binds to integrins, e.g. which comprises RGD peptides, or the D-β-Edipeptide, which binds to PSMA (prostate-specific membrane antigen), iscoupled to the N-terminus of a tissue factor polypeptide. Although anamidolytic and proteolytic effect was demonstrated in vitro, theconstructs, even in combination with factor VIIa, only displayedextremely weak anti-tumor effect in vivo. The animals only survivedlonger in combination with doxycycline.

Hu et al. (46) describe various fusion proteins and use thereof for theproduction of thromboses in tumor vessels, including a fusion proteinfrom an oligopeptide with 9 amino acids, containing the RGD sequence,which was coupled to the truncated form of the tissue factor. Again, theRGD peptides were linked to the N-terminus of tTF to obtain RGD-tTF.Functional analysis showed that the fusion protein containing RGD didnot produce any significant inhibition of tumor growth.

The constructs known in the state of the art were thus constructed insuch a manner that the selective binding domain was linked to theN-terminus of the tissue factor polypeptide. It was even emphasized thatthis structure must be chosen because the N-terminus, on the basis ofstructural models, was considered to be an especially favorable site forlinkage, which would not inhibit the initiation of thrombosis.

SUMMARY OF THE INVENTION

In view of this prior art, the problem therefore resides in providingalternative thrombogenic substances, which can effectively inhibit tumorgrowth in vivo.

This problem is now solved by fusion polypeptides, which comprise apeptide of 3-30 amino acids, which permits the fusion polypeptide to bebound selectively to tumor vessel endothelial cells, and the tissuefactor (TF) or a fragment thereof, the tissue factor and the fragmentbeing characterized in that they are able to activate blood clottingwhen the fusion polypeptide binds to tumor vessel endothelial cells,these peptides being coupled to one another either directly or via alinker having up to 15 amino acids. The peptide, which enables thefusion polypeptide to be bound selectively to tumor vessel endothelialcells, is coupled to the C-terminus of the peptide, which can activateblood clotting when the fusion polypeptide binds to tumor vesselendothelial cells. The present invention further relates topharmaceutical compositions containing corresponding fusionpolypeptides, and use thereof for the treatment of tumors.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic presentation of binding of the tTF-RGD and tTF-NGRfusion proteins to α_(v)β₃ and CD13. Tumor selectivity is achieved owingto the specificity of the RGD sequence for α_(v)β₃-integrin and of theNGR sequence for CD13 (aminopeptidase N). These receptors are expressedselectively and specifically at high density on tumor endothelial cells,but not on resting endothelial cells in normal tissue (apart from a fewexceptions). The representation of the fusion proteins is highlyschematic and does not provide any information regarding the primarysequence.

FIG. 2: SDS-PAGE and Western Blot analysis of recombinant tTF₁₋₂₁₈ (SEQID NO:2) and tTF fusion proteins. The purity of the tTF and of the tTFfusion proteins was checked by SDS-PAGE and staining with Coomassie Blueafter extraction from E. coli (BL21 DE3) and refolding over a linearurea gradient (6M-1 M). Identity of the proteins was verified by Westernblotting using a monoclonal anti-tissue-factor antibody (clone V1C7,American Diagnostics). Loading in the individual lanes: 1=tTF;2=tTF-RGD; 3=tTF-NGR; 4=tTF-cycloNGR1 (SEQ ID NO:6); 5=tTF-cycloNGR2(SEQ ID NO:7); 6=tTF-cycloNGR3 (SEQ ID NO: 8); 7=tTF-GALNGRSHAG (SEQ IDNO: 5); M=molecular weight marker.

FIG. 3: Determination of the Michaelis constants (Km) for the activationof FX by FVIIa/tTF₁₋₂₁₈ or FVIIa/tTF₁₋₂₁₈ fusion proteins. Theparameters of Michaelis-Menten kinetics were calculated using the methoddescribed by Ruf (45).

FIG. 4: Binding of tTF, tTF-RGD and tTF-NGR to integrin α_(v)β₃. Thebinding of 0.1 μM tTF, tTF-RGD and tTF-NGR to immobilized α_(v)β₃ wasquantified with a polyclonal antibody against human TF (AmericanDiagnostica) in an ELISA. The results are presented as median andinterquartile range. The differences in binding between tTF-RGD and tTFor between tTF-NGR and tTF were statistically significant (p<0.001,Mann-Whitney test).

FIG. 5: Specificity of the binding of tTF-RGD to integrin α_(v)β₃ Thebinding of tTF-RGD (0.1 μM) to immobilized α_(v)β₃ was inhibitedsignificantly by competitive inhibition with the synthetic peptideGRGDSP (SEQ ID NO:33) (1-10 μM) (p<0.001, Mann-Whitney test for both RGDpeptide concentrations).

FIG. 6: Binding of tTF and tTF-RGD to human endothelial cells. A: FACSanalysis of endothelial cells incubated with 0.1 μM tTF (2) or with 0.1μM tTF-RGD (3) for 60 min at 4° C. B: A 75% reduction in binding wasdemonstrated by competitive inhibition of the tTF-RGD fusion proteinwith 1 μM GRGDSP (SEQ ID NO:33) (4). Curves 1 in A and B show thenegative control.

FIG. 7: Inhibition of a human lung carcinoma (CCL185) growing as axenograft in athymic nude mice by intravenous therapy with tTF fusionproteins (tTF-RGD, n=6; tTF-NGR, n=6) compared with tumor growth withinfusion of physiological saline solution (NaCl, n=8) or tTF (n=1). Thevertical arrows indicate the times of injection with the respectivesubstances.

FIG. 8: Inhibition and partial remission of a human malignant melanoma(M21) growing as a xenograft in athymic nude mice by intravenous therapywith tTF fusion proteins (tTF-RGD, n=3; tTF-NGR, n=3) compared withtumor growth with infusion of physiological saline solution (NaCl, n=4)or tTF (n=4). The vertical arrows indicate the times of injection withthe respective substances.

FIG. 9: Macroscopic in vivo photograph of a tumor-bearing mouse 20 minafter injection of the tTF-NGR fusion protein (A, left half of thepicture) or NaCl (A, right half of the picture). The macroscopic picturewith bluish-livid coloration of the tumor after injection of tTF-NGR isindicative of tumor necrosis. After 60 min, the two mice wereexsanguinated, the tumor was excised in toto and examinedhistologically. In B, we can see the hemorrhagic imbibition of the tumortreated with tTF-NGR as a sign of secondary hemorrhage as a result ofincipient tumor necrosis. In contrast, the NaCl-treated tumor appears tobe vital (C).

FIG. 10: Histology of the melanoma tumor 1 hour after intravenousinjection of tTF-RGD (A and B), tTF-NGR (C and D) and common salt (E andF) in the caudal vein of the tumor-bearing nude mouse. In the tumorstreated with the tTF fusion proteins, the blood vessels appear to bethrombolytically occluded (arrows). Extensive tumor necroses areobservable in the supply region of the vessel occluded by a blood clot(A-D). The photographs are of representative areas of the tumors (A, Cand E: 200× magnification, B, D and F 400× magnification; HE staining(staining described e.g. in H. C. Burck, Histologische Technik-Leitfadenfür die Herstellung mikroskopischer Präparate in Unterricht und Praxis,5th edition, Thieme Verlag, Stuttgart 1982, pages 109 ff.).

FIG. 11: Representative histologies of heart (A), kidney (B), liver (C)and lung (D) 1 hour after injection of 4 mg/kg BW tTF-NGR. Thromboses ornecroses were not detected microscopically in any of these organs. (HEstaining; 200× magnification).

FIG. 12: Amino acid sequence of human tissue factor (TF) (SEQ ID NO:1).

FIG. 13: Amino acid sequence of the truncated human tissue factortTF₁₋₂₁₈ (SEQ ID NO:2) (also designated tTF for short within the scopeof the present application).

FIG. 14: Amino acid sequence of the fusion polypeptide tTF-GRGDSP (SEQID NO:3) (also abbreviated to tTF-RGD).

FIG. 15: Amino acid sequence of the fusion polypeptide tTF-GNGRAHA (SEQID NO:4) (also abbreviated to tTF-NGR).

FIG. 16: Amino acid sequence of the fusion polypeptide tTF-GALNGRSHAG(SEQ ID NO:5).

FIG. 17: Amino acid sequence of the fusion polypeptide tTF-GCNGRCG (SEQID NO:6) (also abbreviated to tTF-cycloNGR1).

FIG. 18: Amino acid sequence of the fusion polypeptidetTF-GCNGRCVSGCAGRC (SEQ ID NO:7) (also abbreviated to tTF-cycloNGR2).

FIG. 19: Amino acid sequence of the fusion polypeptide tTF-GCVLNGRMEC(SEQ ID NO:8) (also abbreviated to tTF-cycloNGR3).

FIG. 20: Nucleotide sequence of the truncated human tissue factortTF₁₋₂₁₈ (SEQ ID NO:9) (also designated tTF for short within the scopeof the present application).

FIG. 21: Nucleotide sequence of the fusion polypeptide tTF-GRGDSP (SEQID NO:10) (also abbreviated to tTF-RGD).

FIG. 22: Nucleotide sequence of the fusion polypeptide tTF-GNGRAHA (SEQID NO:11) (also abbreviated to tTF-NGR).

FIG. 23: Nucleotide sequence of the fusion polypeptide tTF-GALNGRSHAG(SEQ ID NO:12).

FIG. 24: Nucleotide sequence of the fusion polypeptide tTF-GCNGRCG (SEQID NO:13) (also abbreviated to tTF-cycloNGR1).

FIG. 25: Nucleotide sequence of the fusion polypeptidetTF-GCNGRCVSGCAGRC (SEQ ID NO:14) (also abbreviated to tTF-cycloNGR2).

FIG. 26: Nucleotide sequence of the fusion polypeptide tTF-GCVLNGRMEC(SEQ ID NO:15) (also abbreviated to tTF-cycloNGR3).

FIG. 27: Nucleotide sequence of the oligonucleotides for production oftTF₁₋₂₁₈ (SEQ ID NO:2).

A: 5′-primer (SEQ ID NO:16); B: 3′-primer (SEQ ID NO:17).

FIG. 28: Nucleotide sequence of the oligonucleotides for production oftTF-GRGDSP (SEQ ID NO:3).

A: 5′-primer (SEQ ID NO:18); B: 3′-primer (SEQ ID NO:19).

FIG. 29: Nucleotide sequence of the oligonucleotides for production oftTF-GNGRAHA (SEQ ID NO:4).

A: 5′-primer (SEQ ID NO:20); B: 3′-primer (SEQ ID NO:21).

FIG. 30: Nucleotide sequence of the oligonucleotides for production oftTF-GCNGRCG (SEQ ID NO:6).

A: 5′-primer (SEQ ID NO:22); B: 3′-primer (SEQ ID NO:23).

FIG. 31: Nucleotide sequence of the oligonucleotides for production oftTF-GCNGRCVSGCAGRC (SEQ ID NO:7). A: 5′-primer (SEQ ID NO:24); B:3′-primer (SEQ ID NO:25).

FIG. 32: Nucleotide sequence of the oligonucleotides for production oftTF-GCVLNGRMEC (SEQ ID NO:8).

A: 5′-primer (SEQ ID NO:26); B: 3′-primer (SEQ ID NO:27).

FIG. 33: Nucleotide sequence of the oligonucleotides for production oftTF-GALNGRSHAG (SEQ ID NO:5).

A: 5′-primer (SEQ ID NO:28); B: 3′-primer (SEQ ID NO:29).

FIG. 34: a: Inhibition and partial remission of a human malignantmelanoma (M21) growing as a xenograft in athymic nude mice byintravenous therapy with tTF fusion proteins (tTF-RGD, n=7) comparedwith the growth of the tumors with infusion of physiological salinesolution (NaCl, n=9) or tTF (n=11). The vertical arrows indicate thetimes of the injections with the respective substances.

b: Inhibition of a human fibrosarcoma (HT1080) growing as a xenograft inathymic nude mice by intravenous therapy with tTF fusion proteins(tTF-RGD, n=12) compared with the growth of the tumors with infusion ofphysiological saline solution (NaCl, n=15) or tTF (n=14). The verticalarrows indicate the times of the injections with the respectivesubstances.

c: Inhibition of a human lung carcinoma (CCL185) growing as a xenograftin athymic nude mice by intravenous therapy with tTF fusion proteins(tTF-RGD, n=11) compared with the growth of the tumors in infusion ofphysiological saline solution (NaCl, n=10) or tTF (n=5). The verticalarrows indicate the times of the injections with the respectivesubstances. Statistical significance was investigated in each case withthe Mann-Whitney test for independent groups, P values under 0.05 beingregarded as significant. * shows the statistical significance of thedifference between tTF-RGD and buffer.

FIG. 35: Macrograph of a mouse bearing an M21 tumor at the end oftreatment (day 7) with tTF-RGD fusion protein (A, C) or NaCl (B, D). Thedifference in size and the different appearance of the tTF-RGD-treatedtumors, which in contrast to the apparently vital control tumor showclear signs of necrosis, are readily discernible.

FIG. 36: H-E staining of tumors and organs of mice treated with tTF-RGDand physiological saline solution

Severe thrombosis and necrosis of tumor cells was observed in animalstreated with tTF-RGD (A: 200×, B: 400×). Arrows show examples ofthromboses in blood vessels of the tumor. No obvious thrombosis ornecrosis occurred in animals treated with saline (C: 200×, D: 400×).Arrows show intact blood vessels of the tumor with some erythrocytes.Heart (E), lung (F), liver (G) and kidney (H) of the animals treatedwith tTF-RGD did not show any visible thrombosis or necrosis.

FIG. 37: Action of tTF-NGR in a fibrosarcoma model

Mice bearing a fibrosarcoma (HT1080) were investigated by magneticresonance imaging (MRI) without (pre tTF-NGR) and 6 hours after (posttTF-NGR) i.v. administration of tTF-NGR. The high or low vascular volumefraction is shown.

DETAILED DESCRIPTION OF THE INVENTION

The problems observed in the prior art were now overcome by fusionpolypeptides, which comprise the following peptides:

a) a peptide of 3 to 30 amino acids capable of selectively binding thefusion polypeptide to tumor vessel endothelial cells; and

b) a tissue factor (TF) or a fragment thereof, the tissue factor and thefragment being characterized in that they are able to activate bloodclotting when the fusion polypeptide binds to tumor vessel endothelialcells,

wherein the peptides a) and b) are coupled to one another eitherdirectly or via a linker having up to 15 amino acids, characterized inthat the peptide capable of selectively binding the fusion polypeptideto tumor vessel endothelial cells is coupled to the C-terminus of thepeptide capable of activating blood clotting upon binding of the fusionpolypeptide to tumor vessel endothelial cells. The present inventionfurther relates to drugs containing corresponding fusion polypeptidesand the use thereof for the treatment of tumors.

In addition to sequences a) and b), the fusion polypeptides according tothe invention may comprise additional sequences, provided these do nothave an adverse effect on the steric conformation of the fusionpolypeptide and do not hamper the formation of the enzyme-substratecomplex that triggers blood clotting. The fusion polypeptides accordingto the invention may for example contain sequences of a His-Tag, whichsimplify the recombinant expression and purification of the peptide (cf.Examples). The presence of these sequences is not necessary, however.According to a preferred embodiment of the invention, the fusionpolypeptide therefore comprises:

a) a peptide of 3 to 30 amino acids capable of selectively binding thefusion polypeptide to tumor vessel endothelial cells; and

b) a tissue factor (TF) or a fragment thereof, the tissue factor and thefragment being characterized in that they are able to activate bloodclotting when the fusion polypeptide binds to tumor vessel endothelialcells,

wherein the peptides a) and b) are coupled to one another eitherdirectly or via a linker having up to 15 amino acids. According to aparticularly preferred embodiment of the invention, the fusionpolypeptide comprises:

a) a peptide of 3 to 30 amino acids capable of selectively binding thefusion polypeptide to tumor vessel endothelial cells; and

b) a tissue factor (TF) or a fragment thereof, the tissue factor and thefragment being characterized in that they are able to activate bloodclotting when the fusion polypeptide binds to tumor vessel endothelialcells,

wherein the peptides a) and b) are coupled to one another.

According to the invention it was surprisingly shown, that fusionpolypeptides from a particularly small peptide capable of selectivelybinding the fusion polypeptide to tumor vessel endothelial cells, and apeptide capable of activating blood clotting when the fusion polypeptidebinds to tumor vessel endothelial cells, are especially advantageous forantivascular tumor therapy. The small size of the polypeptide whichpermits binding to tumor vessel endothelial cells improves theorientation of the fusion protein to the phospholipid membrane of theendothelial cell. Formation of the enzyme/substrate complex that isessential for blood clotting is not sterically hindered and the tissuefactor TF, which can activate blood clotting, is not subjected to achange of conformation.

According to a preferred embodiment of the present invention, thepeptide capable of activating blood clotting when the fusion polypeptidebinds to tumor vessel endothelial cells is the tissue factor TF with theamino acid sequence shown in SEQ ID NO:1 (FIG. 12). The inventionfurther comprises tissue factor sequences having an amino acid homologyof at least 70% or at least 80% to SEQ ID NO:1 (FIG. 12), sequences witha homology of at least 95% being especially preferred. The degree ofhomology is determined by writing the two sequences one above the other,four gaps on a length of 100 amino acids being possible, to achievemaximum possible agreement of the sequences being compared (cf. Dayhoff,Atlas of Protein Sequence and Structure, 5, 124, 1972). Then thepercentage of the amino acid residues of the shorter of the two aminoacid chains is determined, which stands opposite to identical amino acidresidues on the other chain.

The peptide capable of activating blood clotting in tumor vessels whenthe fusion polypeptide binds to tumor vessel endothelial cells canmoreover be a fragment of the tissue factor TF or a fragment of asequence homologous to TF. Preferably the fragment has the sequenceshown in SEQ ID NO:2 (FIG. 13). The sequence (tTF₁₋₂₁₈ or tTF for short)shown in SEQ ID NO:2 (FIG. 13) comprises the N-terminal 218 amino acidsof TF. Moreover, according to the invention it is also possible to usefragments of tTF that lack several amino acids at the N-terminus orC-terminus, relative to tTF. For example, it is possible to usefragments that lack up to 10 amino acids at the N-terminus (tTF₁₁₋₂₁₈).Furthermore, fragments can be used that lack up to 8 amino acids at theC-terminus (tTF₁₋₂₁₀), such as (tTF₁₋₂₁₄).

The present invention relates to fusion polypeptides wherein the peptidecapable of selectively binding to the endothelial cells of tumor vesselsis coupled to the C-terminus of the peptide capable of activating bloodclotting. According to the invention, the term “tumor vessel endothelialcells” and the term “endothelial cells in tumor vessels” are used torefer to cells covering the blood vessels of a tumor. It was establishedaccording to the invention that the above arrangement ensuresorientation of the fusion protein perpendicularly to the phospholipidmembrane of the endothelial cell, which is especially advantageous fortriggering blood clotting. This orientation corresponds to the naturalorientation of the TF during induction of blood clotting. As shown inFIG. 3, very similar Michaelis-Menten kinetics are found with respect toactivation of factor X by FVIIa/tTF₁₋₂₁₈ or FVIIa/tTF₁₋₂₁₈ fusionproteins for all constructs produced in this way. In the prior art, incontrast, the peptide which activates clotting was coupled to theC-terminus of the targeting molecule (cf. (16)). The fusion polypeptidesaccording to the invention thus differ fundamentally from the peptidesused in the prior art.

The peptide capable of selectively binding the fusion polypeptide totumor vessel endothelial cells can be any peptide which has a length of3-30 amino acids and binds tumor vessel endothelial cells with highspecificity. Corresponding peptides can be isolated from peptidelibraries by methods that are usual in the state of the art. They canhave a linear or cyclic structure, depending on the peptide library thatis chosen.

According to one embodiment of the present invention, the peptides thatpermit selective binding of the fusion polypeptide to tumor vesselendothelial cells comprise the amino acid sequence RGD or NGR. Bothsequences were known in the prior art for their specific binding tointegrins, especially α_(v)β₃ and α_(v)β₅ integrins (RGD peptides), andas cell adhesion motifs (NGR peptides) (cf. (8)). According to theinvention it was shown, surprisingly, that these peptides are especiallysuitable to be part of a fusion polypeptide, the other part of which isa peptide capable of activating blood clotting in tumors when the fusionpolypeptide binds to tumor vessel endothelial cells.

Especially advantageous effects were obtained with the linear peptideswith the sequences GRGDSP (SEQ ID NO:33), GNGRAHA (SEQ ID NO:34) andGALNGRSHAG (SEQ ID NO:35) and the cyclic peptides with the sequencesGCNGRCG (SEQ ID NO:36), GCNGRCVSGCAGRC (SEQ ID NO:37) and GCVLNGRMEC(SEQ ID NO:38). It was demonstrated that fusion polypeptides comprisingthese sequences and the sequence of the first 218 amino acids of humanTF are highly suitable for antivascular tumor therapy. In particular, itwas shown that these fusion polypeptides cause significant inhibition oftumor growth or reduce the size of tumors (see FIGS. 7 and 8). Theobserved induction of partial remission of the tumors (cf. FIG. 8)points to anticipation of positive results in human tumor therapy basedon the high predictive power of the mouse model (42, 43, 44).

The invention further comprises fusion proteins with cyclic RGDpeptides, since cyclization improves the affinity for integrins (asdescribed for example in reference 21).

The present invention further relates to fusion polypeptides having oneof the sequences shown in SEQ ID NO:3-8 (FIG. 14-19).

According to another embodiment, the present invention relates tonucleic acids encoding a fusion polypeptide, as described above.Corresponding nucleic acids can, for example, have one of the sequencesshown in SEQ ID NO:10-15 (FIG. 21-26).

In yet another aspect, the present invention relates to vectorscomprising one of the aforementioned nucleic acids. Correspondingvectors usually also comprise regulatory sequences for expression of thenucleic acid. Said vectors are comprehensively described in the priorart and are available commercially from a large number of companies.

In a further embodiment, the present invention relates to cellscomprising one of the aforesaid nucleic acids or vectors. The cells aregenerally used for expression of the nucleic acid and recombinantproduction of the fusion polypeptides according to the invention. Alarge number of cells may find application for this purpose, includingE. coli, yeast cells and animal cell lines, such as CHO- or COS-cells.Appropriate cells and the use thereof are described comprehensively inthe prior art.

The polypeptides of the invention according to claim 1 can further beproduced by other suitable methods, for example by chemical coupling ofindividual peptides. Thus, individual peptides can be produced bymethods that are conventional in the state of the art, e.g. by chemicalsynthesis or by heterologous expression, and are then joined together bycoupling.

Finally, the present invention also relates to pharmaceuticalcompositions comprising the fusion polypeptides, nucleic acids, vectorsor cells described above. The pharmaceutical compositions may furthercomprise pharmaceutically compatible carriers, excipients or adjuvants.Moreover, the polypeptides in said pharmaceutical composition may bepresent in a modified state, e.g. pegylated, i.e. coupled to apolyethylene glycol molecule.

The fusion polypeptides according to the invention or pharmaceuticalcompositions containing these fusion polypeptides may be used for thetreatment of neoplastic diseases, and especially for antivascular tumortherapy. Neoplastic diseases that may be considered for treatment withthe aid of the fusion polypeptides according to the invention orpharmaceutical compositions containing these fusion polypeptides includefor example bronchial carcinomas and other tumors of the thorax andmediastinum, breast cancers and other gynecological tumors, colorectalcarcinomas, pancreatic carcinomas and other tumors of thegastrointestinal tract, malignant melanomas and other skin tumors,tumors in the head and neck region, prostate carcinomas and otherurogenital tumors, sarcomas, endocrine-active tumors, leukemias andMyelodysplastic Syndromes and Hodgkin lymphomas and non-Hodgkinlymphomas.

Further, benign tumors, for example hemangiomas, and neovascularizationin diabetic retinopathy, can also be treated.

Apart from intravenous administration, subcutaneous and intraperitonealadministration of the fusion polypeptides or pharmaceutical compositionsis also possible. By packaging in pharmaceutical vehicles, which preventcleavage of the fusion polypeptides in the gastrointestinal tract, thefusion polypeptides or pharmaceutical compositions may also beadministered orally.

It may further be advantageous to combine administration of the fusionpolypeptides according to the invention with other therapeuticapproaches, e.g. cytotoxic chemotherapy or irradiation. Combination withother active substances, e.g. combination with factor VIIa ordoxycycline, is also possible, but preferably combination of thepolypeptide according to the invention with factor VIIa or doxycyclineis not necessary.

The invention is described in more detail on the basis of the followingexamples:

EXAMPLES Example 1 Expression and Purification of tTF and tTF FusionProteins

The cDNA coding for the N-terminal 218 amino acids of tissue factor TF(designated as tTF hereinafter) was synthesized by the polymerase chainreaction (PCR) using the primers shown in SEQ ID NO:16 and SEQ ID NO:17(FIG. 27) and cloned into the expression vector pET-30a(+) (Novagen).The recombinant plasmids were transformed in E. coli (BL21), expressedand purified (Qiagen Plasmid Kit).

Along with the truncated tissue factor tTF, tTF peptide fusion proteinswere constructed, wherein the targeting peptides are first bound to thecarboxyl terminal end of the soluble tissue factor tTF. The followinglinear fusion proteins were constructed:

tTF-GRGDSP (SEQ ID NO:3; FIG. 14; designated tTF-RGD hereinafter; thePCR primers SEQ ID NO:18 and SEQ ID NO:19 (FIG. 28) were used);

tTF-GNGRAHA (SEQ ID NO:4; FIG. 15; designated tTF-NGR hereinafter; thePCR primers SEQ ID NO:20 and SEQ ID NO:21 (FIG. 29) were used);

tTF-GALNGRSHAG (SEQ ID NO:5; FIG. 16; the PCR primers SEQ ID NO:28 andSEQ ID NO:29 (FIG. 33) were used);

In addition, the following cyclic fusion proteins were synthesized:

tTF-GCNGRCG (SEQ ID NO:6; FIG. 17; designated tTF-cycloNGR1 hereinafter;the PCR primers SEQ ID NO:22 and SEQ ID NO:23 (FIG. 30) were used);

tTF-GCNGRCVSGCAGRC (SEQ ID NO:7; FIG. 18; designated tTF-cycloNGR2hereinafter; the PCR primers SEQ ID NO:24 and SEQ ID NO:25 (FIG. 31)were used);

tTF-GCVLNGRMEC (SEQ ID NO:8; FIG. 19; designated tTF-cycloNGR3hereinafter; the PCR primers SEQ ID NO:26 and SEQ ID NO:27 (FIG. 32)were used)

All constructs (including tTF) were expressed in the pET30a(+) vector,which mediates the additional expression of an N-terminal affinity tagof 6 histidine residues and a few vector-coded amino acids. With the aidof this affinity tag, the constructs could be purified by affinitychromatography on a nickel-nitrilotriacetic acid column (Ni-NTA,Novagen). The affinity tag is shown in SEQ ID NO:30. SEQ ID NO:31 andSEQ ID NO:32 show, as examples, the complete amino acid sequences oftTF-GRGDSP with affinity tag (SEQ ID NO:31) and tTF-GNGRAHA withaffinity tag (SEQ ID NO:32).

The constructs were selected so that, on the basis of the known X-raycrystal structure of the tTF:FVIIa complex (19), vertical orientation ofthe tTF fusion protein to the phospholipid membrane of the endothelialcells is ensured, which corresponds to the orientation of the native TF.It was further taken into account that the structure selected should notresult in the tTF causing any steric hindrance to interaction with FVIIaand the macromolecular substrate FX. Owing to the specificity of the RGDsequence for the α_(v)β₃ integrin and of the NGR sequence for CD13(aminopeptidase N), tumor selectivity is achieved, as these receptorsare expressed selectively and specifically at high density on tumorendothelial cells but, apart from a few exceptions, not on restingendothelial cells in normal tissue (see FIG. 1).

tTF and the fusion proteins described tTF-RGD, tTF-NGR, tTF-GALNGRSHAG(SEQ ID NO:5) and tTF-cycloNGR1-3 were transformed and expressed in E.coli (BL21) by means of pET30a(+). Transformed, IPTG-induced E. coliBL21 DE3 were centrifuged and absorbed in 5-7 ml lysis buffer (10 mMTris-HCl, pH 7.5; 150 mM NaCl; 1 mM MgCl₂; 10 μg/ml aprotinin; 2 mg/mllysozyme)/g pellet and 20 μl Benzonase (Novagen) added. After 90 minincubation at room temperature (RT) and centrifugation at 12 000 g, 20min, 4° C., the pellet was resuspended and homogenized by ultrasonictreatment in washing buffer (10 mM Tris/HCl, pH 7.5; 1 mM EDTA; 3%Triton X-100). Inclusion bodies were dissolved over night at RT in 2-4ml/g pellet on denaturing buffer (6 M guanidinium chloride, 0.5 M NaCl,20 mM NaH₂PO₄, 1 mM DTT). The supernatant from centrifugation (5000 g,30 min, 4° C.) was filtered with a 0.22 μg filter. The constructs werepurified until homogeneous on a nickel-nitrilotriacetic acid column(Ni-NTA, Novagen) via the additionally introduced His-Tag sequences ofthe construct. Purification and folding of the proteins were carried outwith the His Bind Buffer Kit (Novagen). This was followed by dialysisagainst TBS buffer (20 mM Tris, 150 mM NaCl, pH 7.4).

The identity of the proteins was confirmed by SDS-PAGE, Western Blot andmass spectroscopy (see FIG. 2).

Example 2 Functional Characterization of tTF and tTF Fusion Proteins

The functional activity of these fusion proteins with respect tocofactor activity in the activation of factor X to factor Xa via factorVIIa was demonstrated in vitro by Michaelis-Menten analyses. The abilityof tTF and of the tTF fusion polypeptides to intensify the specificproteolytic activation of FX via FVIIa in the presence of phospholipidswas determined in a slightly modified version of the method described byRuf (45). For this, 20 μl of each of the following reagents was pipettedin microtiter plates: (a) 50 nM recombinant FVIIa (Novo-Nordisk) inTBS-BSA; (b) 0.16 nM-1.6 μM tTF/tTF fusion polypeptide in TBS-BSA; (c)25 mM CaCl₂ and 500 μM phospholipid vesicle(phosphatidylcholine/phosphatidylserine, 70/30, M/M; Sigma). After 10min incubation at room temperature, 20 μl of the natural substrate FX(Enzyme Research Laboratories) was added at a concentration of 5 μM.Then a sample was taken by pipette at one-minute intervals and thereaction was stopped by adding 100 mM EDTA solution. The amount of FXathat formed was measured by addition of the chromogenic substrateSpectrozyme FXa in a Microplate Reader by determining the change inabsorption at 405 nm and the parameters for the Michaelis-Mentenkinetics were analysed by the method described by Ruf. The results showthat both tTF and the tTF fusion polypeptides are functionally activeunder these conditions (FIG. 3). The Michaelis constants (Km) found forthe fusion polypeptides were in the range 0.12-1.2 nM (FIG. 3), and thusin the lower range that is published for tTF. It can therefore beassumed that the functional activity is unaffected by the fusing of tTFwith the peptides.

Example 3 Binding of the tTF Fusion Proteins to α_(v)β₃ In Vitro and InVivo

Binding of tTF-RGD and tTF-NGR to the α_(v)β₃ integrin was demonstratedin an ELISA (Enzyme Linked Immunosorbent Assay), by immobilizingpurified α_(v)β₃ on microtiter plates (see FIG. 4). The specificity ofthe binding of tTF-RGD to α_(v)β₃ was emphasized by the fact that thesynthetic peptide with the sequence GRGDSP (SEQ ID NO:33) (from thecompany Gibco) competitively inhibits the binding of tTF-RGD to α_(v)β₃in this test system (see FIG. 5).

Next the specific binding of tTF-RGD to α_(v)β₃ on endothelial cells wasevaluated. For this, the differential binding of biotinylated tTF andtTF-RGD to endothelial cells in suspension was analysed by FACS(Fluorescence Activated Cell Sorting). The fact that all endothelialcells held in tissue culture are activated, i.e. express α_(v)β₃molecules, is utilized experimentally. This can be detected by variousimmunohistochemical methods. A cultivated endothelial cell thuscorresponds, in relation to its expression pattern with respect toα_(v)β₃ to a tumor endothelial cell. Accordingly, a cultivatedendothelial cell can be used as a model system for the specific bindingof substances to tumor endothelial cells and also permits predictions tobe made concerning the expected toxicity.

Streptavidin-phycoerythrin was used as the detection method. Themeasured fluorescence intensity for tTF-RGD was higher by a factor of 8than for tTF (FIG. 6A). Furthermore, the binding of 0.1 μM tTF-RGD toendothelial cells was lowered competitively by 75% by the administrationof 1 μM of the synthetic peptide GRGDSP (SEQ ID NO:33) (FIG. 6B). Thisemphasizes the specificity of the binding of tTF-RGD to RGD-bindingreceptors on the endothelial cell surface like α_(v)β₃.

Example 4 Antitumor Effects of the tTF Fusion Proteins in an AnimalModel

The tTF-RGD and tTF-NGR fusion proteins were evaluated with respect totheir effects and side-effects on xenografts of human tumors in athymicnude mice. The models established in our laboratory were used for this(33, 34). The cell lines CCL185 (human adenocarcinoma of the lung) andM-21 (human melanoma) were injected subcutaneously into the flank ofmale BALB/c nude mice (9-12 weeks old). On attaining a tumor volume ofabout 50-100 mm³ (CCL185) or 400-600 mm³ (M−21), the mice were assignedto four groups at random. Group 1 received only physiological salinesolution (NaCl), group 2 tTF, group 3 tTF-RGD, and group 4 tTF-NGR (ineach case 1.5-2.0 mg/kg body weight (BW) of the protein). The injectionswere made in the caudal vein of the animals at intervals of 1-3 days(depending on the growth rate of the particular cell line). Considerabletherapeutic activity of the fusion proteins was observed. The tumors ofthe mice treated with tTF-RGD or tTF-NGR fusion proteins weresignificantly inhibited in their growth or were reduced in size as faras partial remission in comparison with tTF or NaCl (see FIGS. 7 and 8).

To verify the mechanism of action of thrombosis induction in tumorvessels, the following experiment was carried out: the human melanomacell line was injected into the flank of two male BALB/c nude mice. Onattaining a tumor size of approx. 500 mm³, 2.0 mg/kg BW tTF-NGR or NaClwas injected into the caudal vein. FIG. 9A shows an in-vivo macrographof the tumor-bearing mouse 20 min after injection of the tTF-NGR fusionprotein (left half of the picture) or NaCl (right half of the picture).The macroscopic picture with bluish-livid coloration of the tumor afterinjection of tTF-NGR indicates tumor necrosis. After 60 min the micewere exsanguinated, the tumor was excised in toto and investigatedhistologically. FIG. 9B shows the hemorrhagic imbibition of the tumortreated with tTF-NGR as a sign of secondary hemorrhage as a result ofincipient tumor necrosis. In contrast, the tumor treated with NaClappears to be vital (FIG. 9C).

Histological analysis of the melanoma tumor shows microscopicallyvisible thrombus formation in the blood vessels (FIG. 10A-D). Thisfinding verifies the suggested mechanism of anti-tumor effects oftTF-NGR, i.e. induction of thrombi in the blood vessels. The highselectivity of tTF-NGR for tumor blood vessels is demonstrated by theabsence of histological detection of clotting and necrosis in normaltissue such as heart, kidney, liver and lung (FIG. 11A-D). Even repeatedhigh doses of tTF-NGR (4 mg/kg BW) did not lead to any visible clotformation or organ toxicity.

Example 5 Antitumor Effects of the tTF Fusion Proteins in the HT1080Tumor Animal Model

The antitumor activity of the tTF-RGD fusion protein was alsoinvestigated in BALB/c nude mice with fibrosarcomas (HT1080). Thesetumors grow rapidly and are well-vascularized. The results of twoexperiments are presented in Table 2 and FIG. 34. After the secondinjection of tTF-RGD, significant inhibition of growth of the HT1080tumors was observed compared with control groups. This effect lasteduntil the end of the experiment on day 7 (P=0.021 for tTF-RGD relativeto the buffer control (physiological saline solution), P=0.005 fortTF-RGD relative to tTF). As in the earlier experiments, partialregression of tumor volume was observed in this model.

TABLE 1 Effect of tTF-RGD on the growth of M21 tumors in mice Mean tumorvolume (mm³) P relative to P relative to Treatment Day 0 Day 7 buffertTF n Buffer 590 ± 77 994 ± 140 ns 9 tTF 558 ± 47 931 ± 147 ns 11tTF-RGD 585 ± 85 514 ± 81  <0.01 <0.05 7 ns: not significant

TABLE 2 Effect of tTF-RGD on the growth of HT1080 tumors in mice Meantumor P volume (mm³) relative to P relative Treatment Day 0 Day 7 bufferto tTF n Buffer 1671 ± 296 2431 ± 559 ns 15 tTF 1751 ± 269 2335 ± 398 ns14 tTF-RGD 1725 ± 197 1241 ± 122 <0.05 <0.01 12 ns: not significant

TABLE 3 Effect of tTF-RGD on the growth of CCL185 tumors in mice Meantumor volume (mm³) P relative to P relative to Treatment Day 0 Day 7buffer tTF n Buffer 39 ± 3 467 ± 137 ns 9 tTF 44 ± 8 764 ± 148 ns 5tTF-RGD 45 ± 5 130 ± 19  <0.01 <0.01 10 ns: not significant

Other tTF fusion proteins can be constructed without any problem by aperson skilled in the art on the basis of the disclosure of the presentinvention. Potential candidates are the peptides TAASGVRSMH (SEQ IDNO:39) and LTLRWVGLMS (SEQ ID NO:40), which bind to NG 2, the murinehomolog of the human melanoma proteoglycan (12). Expression of NG 2 isrestricted to tumor cells and angiogenic vessels of a tumor (35).Another candidate is the synthetic peptide TTHWGFTL (SEQ ID NO:41),which produces selective and potent inhibition of matrixmetalloproteinase-2 (MMP-2) (13). As the integrin α_(v)β₃ evidently alsobinds MMP-2 in an RGD-independent manner, this means that the activeenzyme is localized on the surface of the angiogenic blood vessels (36).A construct consisting of tTF and this MMP-2 inhibitory peptide mightsimilarly mediate the selective binding of tTF₁₋₂₁₈ to the endothelialcell membrane of tumor vessels.

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1. A fusion polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 3, 4, 6, 7, and
 8. 2. A fusionpolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 3, 4, 6, 7, and
 8. 3. A nucleic acidcomprising a nucleotide sequence encoding the fusion polypeptide ofclaim 1 or
 2. 4. A vector comprising the nucleic acid of claim
 3. 5. Anisolated host cell comprising the vector of claim
 4. 6. A compositioncomprising the vector of claim 5 and a pharmaceutically acceptablecarrier, excipient, or adjuvant.
 7. A composition comprising the hostcell of claim 4 and a pharmaceutically acceptable carrier, excipient, oradjuvant.
 8. An isolated host cell comprising the nucleic acid of claim3.
 9. A composition comprising the nucleic acid of claim 3 and apharmaceutically acceptable carrier, excipient, or adjuvant.
 10. Anucleic acid consisting of a nucleotide sequence encoding the fusionpolypeptide of claim 1 or
 2. 11. A composition comprising the fusionpolypeptide of claim 1 or 2 and a pharmaceutically acceptable carrier,excipient, or adjuvant.
 12. A nucleic acid comprising a nucleotidesequence selected from the group consisting of SEQ ID NOS: 10, 11, 13,14, and
 15. 13. A nucleic acid consisting of a nucleotide sequenceselected from the group consisting of SEQ ID NOS: 10, 11, 13, 14, and15.
 14. A vector comprising the nucleic acid of claim 12 or
 13. 15. Anisolated host cell comprising the vector of claim
 14. 16. A compositioncomprising the nucleic acid of claim 12 and a pharmaceuticallyacceptable carrier, excipient, or adjuvant.
 17. A method of treating apatient with a neoplastic disease comprising administering to saidpatient a fusion polypeptide of SEQ ID NO: 3 or 4 in an amount effectiveto inhibit tumor growth.
 18. The method according to claim 17, whereinsaid administering is selected from the group consisting of intravenous,subcutaneous, oral, and intraperitoneal administration, wherein saidoral administration is of a gastrointestinal cleavage resistantformulation.
 19. A method of treating a patient with a neoplasticdisease comprising administering to said patient a fusion polypeptide ofSEQ ID NO: 3 or 4 in an amount effective to reduce the size of thetumors in the patient.
 20. The method according to claim 19, whereinsaid administering is selected from the group consisting of intravenous,subcutaneous, oral, and intraperitoneal administration, wherein saidoral administration is of a gastrointestinal cleavage resistantformulation.
 21. A method of treating a patient with a neoplasticdisease comprising administering to said patient a compositioncomprising the fusion polypeptide of SEQ ID NO: 3 or 4 and apharmaceutically acceptable carrier, excipient, or adjuvant in an amounteffective to inhibit tumor growth.
 22. The method according to claim 21,wherein said administering is selected from the group consisting ofintravenous, subcutaneous, oral, and intraperitoneal administration,wherein said oral administration is of a gastrointestinal cleavageresistant formulation.
 23. A method of treating a patient with aneoplastic disease comprising administering to said patient acomposition comprising the fusion polypeptide of SEQ ID NO: 3 or 4 and apharmaceutically acceptable carrier, excipient, or adjuvant in an amounteffective to reduce the size of the tumors in the patient.
 24. Themethod according to claim 23, wherein said administering is selectedfrom the group consisting of intravenous, subcutaneous, oral, andintraperitoneal administration, wherein said oral administration is of agastrointestinal cleavage resistant formulation.